Caged compounds are artificial molecules whose biological activity is controlled by light, usually by photolytic conversion from an inactive to an active form. The term "caged" has become popular because it is brief and pictorial, not because it is accurate. In nearly all useful caged biological molecules to date, simple covalent bond formation masks some feature important for biological recognition. Photochemical cleavage of that single bond releases the active species.
Caged compounds are biologically useful because illumination can be easily controlled in timing, location, and amplitude. Therefore abrupt or localized changes in concentration of active species can be generated with controlled amplitudes. This capability is particularly valuable when rapid mechanical mixing is impractical, for example, on the surface of or inside a more-or-less intact cell, tissue, or protein crystal, or when microscopic spatial gradients are desired. Photolysis of caged compounds is one of the best techniques to examine the fast kinetics or spatial heterogenity of biochemical responses in such systems.
Caged compounds are most commonly designed by modifying a desired biomolecule with a suitable photoremovable protecting or "caging" group. To be useful in biological experiments, the protecting group must satisfy several requirements, at least partially. First, it should render the biomolecule inert to the biological system used. Second, it should release the biomolecule in high yield at sufficient speed by photolysis at wavelengths of light that are non-detrimental to the biological preparation. Third, any photoproducts other than the desired biomolecule should not interact or interfere with the biological system.
Caging groups based on the photoisomerization of 2-nitrobenzyl (ortho-nitro-benzyl) derivatives are the most prevalent in present caged compounds. However, its use in biological systems has been limited because the photoproduct, 2-nitrosobenzaldehyde, may react with the released compound or other components of the preparation. Nevertheless, it has been tested for derivatives of ATP, inositol polyphosphate, calcium chelators, amino acids and proton chelators.
A recent review of caged compounds and identification of many caging groups is "Controlling Cell Chemistry with Caged Compounds", Adams et al., Annu. Rev. Physiol., 1993 55:755-84. Numerous other photolabile caging compounds have been reported in the literature, including benzyl bromides; 1-(.varies.-diazobenzyl)pyrene; N-hydroxy-2-thiopyridone; N-hydroxysuccinimidyl p-azidobenzoate; N-hydroxysuccinimidyl ester of p-azidobenzoylglycine; N-hydroxysuccinimidyl p-benzoylbenzoate; p-pentachlorophenyl ester of p-benzoylbenzoylglycine; (bromomethyl)phenylacetate; 1-peptidyl-5-bromo-7-nitroindoline; as well as aromatic diazo compounds such as 1-(2-nitro-4,5-dimethoxy)phenyl-diazoethane, 1-(2-nitro)phenyl-diazoethane, and 1-(2-nitro-3,4,5,6-tetramethyl-diazoethane.
Caged compounds have been used recently in chemical kinetic investigations of neurotransmitter receptors. Hess, Determination of the Chemical Mechanism of Neurotransmitter Receptor-Mediated Reactions by Rapid Chemical Kinetic Techniques, Biochemistry, 32:989-1000 (1993). Rapid mixing is an experimental problem in investigations of these receptors, which are proteins embedded in the membrane of muscle and nerve cells. The proteins control the transmission of signals between cells of the nervous system, and are implicated in a wide variety of diseases and thus are the targets of many clinically important drugs. Some steps of the receptor-mediated reactions involve the binding of a neurotransmitter to its constituent receptor and the opening of transmembrane channels, and occur in less than a millisecond. Determination of the chemical mechanism of the reactions, which must be investigated in intact cells in the sub-ms time region, has been a central goal in neurobiology for several decades. Recently, chemical kinetic investigations of neurotransmitter receptors in intact BC.sub.3 H1 cells in the .mu.s time region were reported.
The introduction of the .varies.-carboxy-o-nitrobenzyl group for protecting carbamate amino groups allowed the synthesis of caged carbamoylcholine. (Milburn et al., Biochemistry 28:48-55 (1989)) This compound is biologically inert, but can be photolyzed to carbamoylcholine, a well-characterized analogue of acetylcholine, with a t.sub.1/2 of 40 .mu.s. Thus, it became possible to equilibrate receptors on a cell surface with caged carbamoylcholine, liberate carbamoylcholine by a laser pulse within about 120 .mu.s, and investigate the kinetics of the formation of acetylcholine receptor-channels on a .mu.s time scale. However, photolysis of analogous N-caged derivatives of gamma-aminobutyric acid (GABA) and glycine which activate specific inhibitory receptors in the central nervous system occurs in the ms time region and with low product quantum yield (&lt;0.05).
Kuzmic et al., Collect. Czech. Chem. Commun., 51:1293-1300 (1986) contains a report of the photochemical hydrolysis of a series of nitrophenyl acetates.
The desire to make chemical kinetic measurements in the .mu.s-to-ms time region with receptors for these central nervous system neurotransmitters required more suitable caged compounds and is an object of this invention.
In particular, it is an object of this invention to produce a photosensitive blocking group for the carboxyl function of amino acids, e.g. glycine, aspartic acid, beta-alanine and .gamma.-aminobutyric acid (GABA), and the excitatory neurotransmitters, e.g. glutamic acid and N-methyl-D-aspartic acid (lVMDA).